www.nature.com/cr www.cell-research.com ARTICLE OPEN DNA damage triggers tubular endoplasmic reticulum extension to promote apoptosis by facilitating ER-mitochondria signaling Pengli Zheng1,2, Qingzhou Chen1, Xiaoyu Tian1, Nannan Qian1,3, Peiyuan Chai1, Bing Liu1,4, Junjie Hu 5,6, Craig Blackstone2, Desheng Zhu7, Junlin Teng 1 and Jianguo Chen1,4 The endoplasmic reticulum (ER) is composed of the nuclear envelope, perinuclear sheets and a peripheral tubular network. The peripheral ER and mitochondria form tight contacts at specific subdomains, which coordinate the functions of the two organelles and are required for multiple cellular processes such as Ca2+ transfer and apoptosis. However, it is largely unknown how ER morphology and ER-mitochondria signaling are dynamically regulated under different physiological or pathological conditions such as DNA damage. Here we show that the peripheral, tubular ER undergoes significant extension in response to DNA damage, and that this process is dependent on p53-mediated transcriptional activation of the ER-shaping proteins REEP1, REEP2 and EI24 (alias PIG8). This promotes the formation of ER-mitochondria contacts through EI24 and the mitochondrial outer membrane protein VDAC2, facilitates Ca2+ transfer from ER to mitochondria and promotes DNA damage-induced apoptosis. Thus, we identify a unique DNA damage response pathway involving alterations in ER morphology, ER-mitochondria signaling, and apoptosis. Cell Research (2018) 28:833–854; https://doi.org/10.1038/s41422-018-0065-z INTRODUCTION transfer with high efficiency from the ER to mitochondria, which The endoplasmic reticulum (ER) is the largest membranous is necessary for mitochondrial metabolism.16 However, dramati- organelle and performs essential roles in protein synthesis and cally increased ER-mitochondria Ca2+ flux triggers apoptosis by secretion, Ca2+ homeostasis, and lipid metabolism. Morphologi- activating the mitochondrial permeability transition pore and cally, the ER consists of the nuclear envelope, high density sheets subsequently releasing cytochrome c.16 Therefore, ER- in the perinuclear region, and a peripheral tubular network.1,2 mitochondria contacts are critical for determining cell fate. A Dysregulation of proper ER morphology is associated with various complex formed by voltage-dependent anion channel 1 (VDAC1), human diseases such as hereditary spastic paraplegia (HSP),3 glucose-regulated protein 75 (GRP75), and the inositol-1,4,5- Alzheimer’s disease4 and cancer.5 Several proteins have been trisphosphate receptor (IP3R), known as the MAM complex,17 identified to regulate ER morphology. Climp63, kinectin and p180 has been reported to be involved in the response to several stress are important for the formation of ER sheets,6 whereas Reticulons conditions, such as ER stress and oxidative stress.17–19 (Rtns),7 receptor expression enhancing proteins (REEPs),8 Atlas- Upon DNA damage, cells initiate several response pathways tins,9 and Lunapark (Lnp1)10 generate the tubular ER. Tubular ER- that include DNA-PK and ATM/ATR to activate DNA repair, cell shaping proteins of the reticulon and REEP families contain one or cycle arrest and/or apoptosis.20 An improper or insufficient DNA more intramembrane hairpin regions consisting of two closely- damage response can lead to genetic mutations and cancer spanned short transmembrane domains that are proposed to development.21 One key player in the DNA damage response is form wedge-like structures within the outer leaflet of the lipid the tumor suppressor p53, which promotes cell cycle arrest and bilayer, stabilizing the high membrane curvature of the ER DNA repair in response to moderate DNA damage, but apoptosis tubules.11 REEP1 and REEP2 (REEP1/2) are both reported to be to severe DNA damage.22 Among the p53 target proteins, HSP-related proteins.8,12 REEP1 also plays important roles in lipid etoposide-induced protein 2.4 (EI24) (alias p53-induced gene 8 droplet formation,13 ER stress response,14 and ER-mitochondria protein, PIG8) is an ER-localized transmembrane protein that was contacts.15 originally reported to be a tumor suppressor23 and is frequently The ER and mitochondria are often tightly associated at specific lost or mutated in various cancers.24–27 EI24 has been reported to subdomains via tethering mediated by mitochondria-associated inhibit cell growth and promote apoptosis.28 Loss of EI24 leads to ER membrane (MAM) proteins. These contacts enable Ca2+ resistance to DNA damage-induced cell death29 and is associated 1Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China; 2Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA; 3College of Life Sciences, Jiangsu Normal University, Xuzhou 221116, China; 4Center for Quantitative Biology, Peking University, Beijing 100871, China; 5National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100871, China; 6Department of Genetics and Cell Biology, College of Life Sciences, Nankai University and Tianjin Key Laboratory of Protein Sciences, Tianjin 300071, China and 7Laboratory Animal Research Center, Peking University, Beijing 100871, China Correspondence: Junlin Teng ([email protected]) or Jianguo Chen ([email protected]) These authors contributed equally: Pengli Zheng, Qingzhou Chen, Xiaoyu Tian. Received: 27 January 2018 Revised: 23 May 2018 Accepted: 13 June 2018 Published online: 20 July 2018 © The Author(s) 2018 DNA damage triggers tubular ER extension P. Zheng et al. 834 with breast tumor invasiveness.30 In addition, in p53-deficient Information, Figure S1c). Indeed, the peripheral/perinuclear ER cells, EI24 acts as an E2F target that contributes to cell survival ratio increased significantly after treatment with DNA damaging after UV irradiation.31 A recent report showed that EI24 associates drugs both in COS7 and U2OS cells (Fig. 1e, f). with the nuclear import machinery and inhibits the nuclear To further confirm the extension of tubular ER upon DNA translocation of p53.32 Thus, the exact role of EI24 in apoptosis damage, we fluorescently co-immunolabeled Rtn4 and Climp63, seems complex. markers of tubular and sheet ER, respectively.6,7 After deconvolu- Although the DNA damage response has been studied tion and 3D-reconstruction, we analyzed the volumetric ratio of intensively over the past few decades, the mechanisms whereby Rtn4-positive objects to Climp63-positive objects, representing DNA damage affects the structures and function of cytoplasmic the volumetric ratio of tubular ER to ER sheets. The volumetric organelles have only begun to be elucidated.33 As ER function is ratio of Rtn4/Climp63 also increased significantly after eto- or cpt- intimately related to the DNA damage response and pathways treatment (Fig. 1g; Supplementary Information, Figure S1e), downstream of p53,34–36 we investigated whether the morphol- confirming that DNA damage induces tubular ER extension. ogy of ER responds to DNA damage. We show that DNA damage We then analyzed protein levels of several ER-localized proteins triggers tubular ER extension via the p53-mediated expression of from various pathways. Of these, the Ca2+ transporter SERCA2, REEP1/2 and EI24, and that this facilitates contacts between ER lipid synthase MOGAT2, and the protein folding chaperone and mitochondria. We further show that under DNA damage calnexin did not increase upon DNA damage whereas CEPT1 conditions, EI24 and VDAC2 mediate the formation of a unique ER- and BiP decreased (Supplementary Information, Figure S1f). Thus, mitochondria contact that promotes mitochondrial Ca2+ uptake the observed tubular ER extension is not due to an increase in and apoptosis. general ER content. To exclude the possibility that ER extension upon DNA damage is a secondary effect of changes in cell shape, we analyzed the RESULTS volume occupied by ER and the whole cell by 3D-reconstruction of DNA damage induces tubular ER extension mCherry-ER and GFP (as an indicator of the whole cell) using 3D To determine whether DNA damage affects ER morphology, we structured illumination microscopy (3D-SIM) (Supplementary assessed changes of the total ER distribution area in various Information, Figure S1g-i). The total cell volume did not increase mammalian cell lines by expressing ER-lumen localized mCherry (Supplementary Information, Figure S1h) whereas the ER volume (mCherry-ER) and treating the cells with the DNA damaging drugs increased significantly (Supplementary Information, Figure S1i). 1234567890();,: etoposide (eto), camptothecin (cpt) or doxorubicin (doxo). In 30 of Moreover, treating cells with the actin-depolymerizing agent 36 mammalian cell lines tested, exposure to DNA damaging drugs cytochalasin B did not block the DNA damage-induced change in dramatically increased the total ER distribution area (Fig. 1a), ER distribution (Supplementary Information, Figure S1j and k). The indicating that this is a common characteristic of the DNA damage polymerization status of microtubules also was not changed after response in mammalian cells. We subsequently focused on COS7 eto treatment (Supplementary Information, Figure S1l). Therefore, and U2OS cells, which are widely used to study ER morphology.9,37 the DNA damage-induced morphological changes of the ER are After eto treatment, the ER -- especially